Abstract The proposed research focuses on development of super-resolution optical imaging methods that utilize reversible and transiently binding probes as the labeling reagents. Using of reversibly-binding probes has the potential of achieving significantly higher labeling density and enhanced spatial resolution. However, currently their application is limited by the lack of suitable labeling reagents towards important cellular targets. Here we propose to expand the repertoire of diffusive labeling tools by developing new labeling methods to facilitate broader usages of PAINT methodology in biological studies and new statistical algorithms to achieve the optimal spatial resolution from localization dataset collected with diffusive labeling. Specifically we will focus on (1) developing imaging probes for phosphotyrosine signaling proteins based on Src- homology 2 (SH2) domains, (2) developing a hybrid genetic-chemical labeling method based on protein-peptide interactions that allows imaging of cellular proteins, and (3) developing statistical models and numerical algorithms to achieve resolution enhancement when target molecules are labeled in multiple rounds with reversible probes.